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First published online 14 September 2005
doi: 10.1242/dev.02037

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Cerebellum- and forebrain-derived stem cells possess intrinsic regional character

¹ Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA
² Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9111, USA

View larger version (163K): [in a new window]   Fig. 1. Cerebellar neurospheres give rise to neurons that resemble distinct cerebellar cell types in vitro. E14.5 (A-E) or adult (F-K) cerebellar neurospheres expressing EGFP were plated on a cerebellar feeder layer and allowed to differentiate. Cells derived from the neurospheres that express the molecular marker indicated are double-labeled and appear yellow, whereas neurosphere-derived cells not expressing the marker examined appear green, while feeder-layer derived cells expressing the analyzed marker appear red. A subpopulation of neurosphere-derived cells gave rise to GABA-ergic neurons (A,F) and can be subdivided into the parvalbumin-expressing interneurons (B,G) and the calbindin-positive Purkinje-like cells (C,H). Neurosphere-derived cells also gave rise to neurons that resemble granule cells, as indicated by their expression of glutamate (D,I) and TAG1 (E,J). The expression of synaptophysin implies that the cells can generate synaptic vesicles in vitro (K).

View larger version (67K): [in a new window]   Fig. 2. Math1-negative progenitors give rise to Math1-expressing cells. (A) Math1EGFP expression is restricted to the external granule layer of the cerebellum at E14.5 and overlaps with endogenous Math1 expression. (B) FACS subdivides the Math1EGFP-negative fraction from the Math1EGFP-positive cells. (C) The unsorted cells, as well as the Math1EGFP-negative cells, give rise to neurospheres, whereas the Math1EGFP-positive cells do not form neurospheres. (D,E) FACS of neurospheres derived from Math1EGFP-negative cells plated on a feeder layer reveals the upregulation of Math1EGFP after 2 days in culture. The perdurance of the EGFP allows the double-labeling of Math1EGFP-positive neurons expressing Tuj-1 (E).

View larger version (67K): [in a new window]   Fig. 3. Forebrain-derived neurospheres use normal migratory routes and give rise to neurons with the characteristics of olfactory bulb granule cells and cortical interneurons when transplanted to the forebrain. Constitutive EGFP-expressing forebrain-derived neurospheres were transplanted into the neonatal forebrain (P4) and analyzed at P21. Neurosphere-derived cells were using normal migratory routes and were found in various regions of the brain (A). GABA-immunopositive cells with the morphology of granule neurons were found in the granule cell layer of the olfactory bulb (B), and neuroblasts utilizing chain-migration were observed within the olfactory stream and expressed Tuj1 (C). GABA-positive cells were found distributed over most layers of the cortex (D). Some cells stayed close to the graft site and were expressing GFAP (E). Scale bars: 1 mm in A; 50 µm in B1,C1,D1,E1; 20 µm in B2,C2,D2,E2. Note that regions shown in boxes B1-E2 represent higher power taken from the approximate regions shown in A and that the two higher power pictures in C1 versus C2, as well as D1 versus D2, are different examples.

View larger version (62K): [in a new window]   Fig. 4. Cerebellar-derived neurospheres mainly give rise to astroglia when transplanted to the forebrain. Constitutive EGFP-expressing adult cerebellar-derived neurospheres were transplanted into the neonatal forebrain (P4) and analyzed at P21. The grafted cells mainly remained in close proximity to the injection site (A). GFAP-expressing cells were found in the corpus callosum (B), in the cortex along the needle tract (C) and close to the ventricle (D). TAG1-expressing cells were found close to the graft site (D). Scale bars: 1 mm in A; 50 µm in B1,C1,D1,E1; 20 µm in B2,C2,D2,E2. Although it appears that many of the GFP cells in B2-D2 are not expressing GFAP, this is a result of the different localization of GFP and GFAP in these cells. Reconstruction of optical stacks demonstrates that the GFP cells shown in these figures are largely GFAP positive.

View larger version (161K): [in a new window]   Fig. 5. Forebrain-derived neurospheres mainly give rise to astroglia when transplanted to the cerebellum. Constitutive EGFP-expressing E14.5 forebrain-derived neurospheres were transplanted into the neonatal cerebellum (P4) and analyzed at P21. The cells integrated into the host tissue and expressed GFAP, displaying morphology of either astrocytes (A-C) or Bergman radial glial cells (D,E, arrows). Only one neurosphere-derived neurofilament-expressing cell was observed (G-I).

View larger version (99K): [in a new window]   Fig. 6. Embryonic and adult-derived cerebellar neurospheres give rise to neurons with the characteristics of GABA-ergic interneurons and granule cells in vivo. Constitutive EGFP-expressing E14.5- and adult-derived cerebellar neurospheres were transplanted into the neonatal cerebellum (P4). A subpopulation of cells was observed to integrate into the host cerebellum and express GABA (A-F), indicating that they are interneurons. EGFP-positive calbindin-expressing cells with the morphology of Purkinje cells were detected in the Purkinje cell layer of E14.5-derived neurospheres, albeit at low frequency (G-I). In adult-derived NSC transplantations, large fusiform cells within the Purkinje cell layer that did not express calbindin were observed (J-L). Most commonly observed were EGFP-expressing cells with the characteristics of granule cells. These possessed a small cell body with three to five protruding dendrites and were immunoreactive for glutamate (M-R). Current-voltage responses of immature (S,U) and mature (T,V) GFP-positive cerebellar granule cells. Immature cells exhibited long-duration, non-repetitive intermediate- and high-threshold calcium spikes, whereas mature granule cells exhibit pronounced inward rectification and fast, repetitive spiking. Synaptic input was not blocked. Step protocol from initial step of –20pA, with 5pA increments. In T, the +10pA step is not shown for clarity. Scale bars: 30 µm in C,F,R; 20 µm in I,F,L. Horizontal bar, 50 milliseconds; vertical bar 10 mV in S,T; horizontal bar, 200 mseconds; vertical bar, 10 mV in U,V.